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| Laser TIRF 3 - The Principle |
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The analysis of images which have been acquired with conventional widefield fluorescence excitation near the cell membrane is difficult due to the background fluorescence from other planes which are not in focus. Only under TIRF conditions a resolution of < 200 nm in z is achieved by selective illumination.
Required resolution
The resolution needed for the purpose is achieved neither by epifluorescence nor by confocal microscopy. The problem is solved by a technique in which fluorochromes are excited within a thin layer only. TIRF microscopy is particularly suited to the task, as the principle allows only those molecules to be excited which are in a layer of typically 100 - 200 nm above the coverslip.
The principle of total internal reflection
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Beam path of incidence at less than the critical angle | Beam path of total reflection |
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Total reflection occurs at the interface between an optically denser and an optically less dense medium, if a light ray is incident at an angle that, according to Snell's law of refraction, is greater than the so-called critical angle (i.e., α2 = 90°).
Hence,
For the transition from the glass coverslip (n1 = 1.518) into water (n2 = 1.33), a critical angle of 61° results. With total reflection, a stationary evanescent (= decaying) wave is formed at the interface between the two media. The intensity of this wave decays exponentially with increasing distance from the interface.
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I(z) = Intensity at a distance z from the interface.
I(0)= Intensity at the interface.
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The penetration depth of the evanescent field is defined as the distance d from the interface at which the energy of the light has dropped to 37% of the original level. This distance depends on the angle of incidence and the wavelength of the light used.
Because of the energy distribution, fluorochromes at a greater distance from the interface are not excited. Therefore, a thin optical section about 100-200 nm thick is produced, yielding a very high signal/background ratio. This results in a marked increase in image contrast and Z resolution. | |
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